EP0380675A1 - Method for making sour-milk products - Google Patents

Abstract

registriert ist. Die Fermentation erfolgt bis zur Herstellung des Endprodukts.The process for the production of sour milk products is that the fermentation of milk or milk products is carried out with the living bacteria, the strain Bacillus subtilis 534 being used as such, which was on 28.03.88 in the Allunions culture collection of the Institute of Biochemistry and Physiology of Microorganisms of the Academy of Sciences of the USSR deposited and under number B-1666

is registered. The fermentation takes place until the end product is produced.

Description

Technical field

The present invention relates to the food industry and, more particularly, to a method for producing sour milk events.

Underlying state of the art

Processes for the production of sour milk events are known and widely used today, according to which an acid alarm clock is added to pasteurized milk or buttermilk, in which the bacteria produce lactic acid. The milk or its components coagulate at a temperature between 28 and 38 ° C within 10 to 24 hours. After quality control and confirmation of the usability of the manufactured product (kefir, acidophilus milk) it is sold. Storage of the product at a temperature between 2 and 8 C within 24 hours is permitted. Lactic acid streptococci, acidophilic lactic acid bacteria and propionic acid bacteria (SU, A, No. 1238746, 1287822, 1271479) are used as acid alarm clocks in the production of the products mentioned.

The sour milk products, which are produced according to the specified methods, have a very weak therapeutic-prophylactic effect in dysbacteria, bacterial infections of the gastrointestinal tract and are even ineffective in purulent inflammatory processes, food allergy and diathesis. Because of the high acidity, the products described are contraindicated in ulcers and gastritis with increased secretion.

In the production of sour milk products according to the specified methods, the contaminated product can be contaminated with the conditionally pathogenic bacterial flora (staphylococci, salmonella, shigella and other bacteria), which leads to spoilage of the product and sometimes to food poisoning. The well-known sour milk products kefir and azodophilus milk have a short storage time (around 24 hours).

Disclosure of the invention

The invention has for its object to ent a method by using a new type of bacteria wrap, which enables to produce sour milk products with the therapeutic - prophylactic effect against bacterial infections, dysbacteria, diathesis and food allergy.

registriert ist.The object is achieved in that in the process according to the invention for the production of sour milk products by fermentation of milk or milk products with the help of the living bacteria with the production of the end product, according to the invention, the strain of bacteria Bacillus subtilis 534 is used as living bacteria, which on 28.03.88 in deposited in the All-Union Culture Collection of the Institute for Biochemistry and Physiology of Microorganisms of the Academy of Sciences of the USSR and under number B-1666

is registered.

For the purpose of fermentation, it is advisable to add the specified strain in an amount of 10 ³ to 10 ¹¹ living cells per 1 dm ³ milk or milk product.

The process according to the invention produces the product which has an effective therapeutic-prophylactic behavior in the case of bacterial infections, dysbacteria, diathesis and food allergy. This is connected with the fact that the new strain actively produces protein antibiotics into the external medium with a broad spectrum of activity, proteolytic ferments and immunomodulators. The bacteria can also penetrate into the tissue of the pathological focus, which results in the healing effects of purulent inflammatory processes outside the gastrointestinal tract. The known milk products were not used for this purpose.

As a result of the high effectiveness of the antibacterial substance excreted by the new strain, the possibility is practically avoided that the pathogenic bacteria, namely staphylococci, streptococci, salmonella, dysentery bacteria, occur in products during the production of storage. In the known methods, the development of the pathogenic bacteria mentioned is not infrequently proven and leads to the spoilage of the product and sometimes to the outbreak of the infection. The one using the new strain Milk products produced can be stored under the same conditions two to five times longer than the products produced by the known methods.

The products produced by the process according to the invention have a pleasant sweet taste.

The best embodiment of the invention

The method according to the invention is carried out as follows.

Milk or milk products (buttermilk, milk serum) are pasteurized at a temperature between 75 and 100 ° C. within 15 to 30 minutes.

You prepare an acid alarm clock. The lyophilized three-day stock culture Bacillus subtilis 534 is used and its suspension in sterile milk is made. The alarm clock is added to the milk or milk products, the temperature of which is 75 to 100 ° C, starting from ₁₀ ³ to ₁₀ ¹¹ living cells per 1 dm ³ . The fermentation takes place at a temperature between 30 and 38 ° C within 6 to 24 hours. After the formation of a uniform, dense clot, the fermentation is completed. A weakly sweet product with no odor is obtained.

The acidity of the manufactured product varies between 20 and 50 ° T. The phosphatase is missing, contamination with a foreign microflora does not occur. The amount of dry residue is 10 to 14% by mass.

The new strain Bacillus subtilis 534 used in the method according to the invention is isolated in humans and is characterized by the following morphological features and physiological properties.

These are chopsticks. Cell size of the one-day agar culture (2.4) × (0.6-0.8) µm. Bacteria mobile, form spores and not capsules, gram-positive. Colonies on meat peptone agar rough, with an uneven edge, with a light pink color, diameter 2 to 12 mm. The strain reproduces at a temperature of 15 to 50 ° C, optimum growth 36 to 37 ° C. Splits glucose, sucrose and mannitol without gas evolution to acid. Does not ferment lactose. Does not form a sweat hydrogen chloride and no indole. Excretes acetylmethyl carbinol and catalase. Sensitive to benzylpenicillin, ampicillin, erythromycin, monomycin, linkomycin, tetracycline, insensitive to polymyxin.

The strain produces the antibiotic with a broad spectrum of activity, which inhibits the growth of staphylococci, streptococci, proteus, pyrocyanic bacteria, salmonella, shigella groups, yeasts, etc. inhibits. He also excretes proteoplytic protein-cleaving enzymes and immunomodulators.

The antagonism of the specified strain against pathogens of the bacterial infection was examined.

The antagonism of the Bacillus subtilis 534 strain against pathogens of bacterial infection was determined using the methodology developed by WINikitenko. Culture tubes containing 5 cm ^{3 of} sterile Kohlbach nutrient broth were inoculated with 40 to 50 million cells each from a test culture (control) and with the same doses of the test culture together with the specified strain. Culture tubes were kept in a thermostat at a temperature of 37 ° C for 24 hours. Since the strain bacteria Bacillus subtilis 534 had an insignificant effect on the density of the broth, the degree of growth inhibition of the test culture was determined with the aid of a photoelectric colorimeter after the difference in boillon in the test and control. The result was considered positive if the density of the misoh culture was 1.2 times or more lower than the boullon density of the test culture.

The Bacillus subtilis 534 strain inhibited the growth of 34 out of 37 examined strains of the staphylococcus, 11 out of 12 strains of the streptococcus, 8 out of 12 strains of the coli bacteria, 7 out of 8 strains of the dysentery bacteria, 7 out of 7 strains of the salmonella group, 6 out of 6 proteus strains, 7 of 8 strains of the Pyozyaneus bacteria. The saprophytic microorganism strains mainly proved to be stable and less sensitive.

In order to demonstrate the production of the antibacterial substances in the external medium, the stock cultures were grown Bacillus su'otilis 534 (3 laboratory series) within 96 hours in Erlenmeyer flasks on a shaking table (240 rpm) at a temperature of 28 ° C on a nutrient medium with the following composition in mass%: pea flour 1.5, sucrose 2, 1, starch 0.85, NaNO ₃ 0.5, CaC0 ₃ 0.5 NaCl 0.5, water everything else. The antibacterial activity of 0.1 cm ³ filtrate of the culture liquid was determined by diffusion in the 2% meat peptone agar. The size of the zones of inhibition when growing test cultures while subtracting the hole diameter (8 mm) was taken into account.

The zones of inhibition for staphylococcus were 24 to 27 mm, for coli bacteria 18 to 22 mm, for Klebsiella 18 to 21 mm, for yeast 22 to 24 mm.

3 rows of the preparation from the living culture Bacillus subtilis 534 were used to test the acute toxicity. Each row was administered 3 intraperitoneally to 3 white mice and 3 white Wistar rats in a dose of 10 or 20 billion cells per 1 cm ^{3 of} 0.9% NaCl solution. All animals stayed alive. They ate good food. The animals were slaughtered on the 3rd, 7th and 14th day of the experiment. The histological examination showed no inflammatory and dystrophic changes in the brain, lungs, kidneys, liver and heart. Enlarged follicles and higher amounts of lymphohistiocytic elements in the red pulp appeared in the spleen.

In order to determine the degree of acute toxicity, 3 series of preparations from the Bacillus subtilis 534 strain were each taken once with the food of 6 white mice and 6 white Wistar rats in a dose of 10 and 20 billion cells. All animals stayed alive. The next day they were slaughtered. In the histological examination of the brain, myocardium, lungs, kidneys, stomach, small and large intestine, the changes compared to control animals (10 mice and 10 rats) were not demonstrated. The liver shows some increase in the amount of lymphohistiocytic infiltrates in the direction of portal tracts. In the spleen there were enlarged follicles and higher amounts of lymphohistiocytic elements in the red pulp and multinucleated Giant cells on.

In the examination of the chronic toxicity, 3 series of preparations from the Bacillus subtilis 534 strain were intraperitoneally administered to 3 white mice and 3 white Wistar rats within 30 days in a dose of 200 million or 1 billion cells per 1 cm ³ 0.9 % NaCl solution injected. Each of the three series of preparations was also taken in the same doses of 6 white mice and 6 white Wistar rats. All animals stayed alive. The weight gain of experimental animals was 11 to 17% (p <0.05) compared to control animals (10 mice and 10 rats). The hair in mice and rats in experimental groups was bright white, soft. The animals were slaughtered on the 31st day. The changes were not detected in the histological examination of the brain, myocardium, kidneys, lungs, stomach, small and large intestine. Lymphohistiocytic infiltrates in the direction of portal channels were found in greater amounts in the liver than in control animals. Lymphoidization of the red pulp and enlarged follicles were observed in the spleens of experimental animals, but the multinucleated giant cells did not appear.

In the course of carrying out experiments aimed at examining chronic toxicity, 23 mice without abnormality were born on the 11th to 21st day in 6 white mice. The latter gave birth to 7 mice without abnormality.

To determine the stability of the strain, 9 capsules of preparation from the Bacillus subtilis 534 strain were stored in sealed bottles at room temperature for 7 years. 9 capsules were stored within 3 months at a temperature of minus 20 to 22 ° C and 9 capsules within 2 months at a temperature of 110 ° C. The amount of living bacteria in capsules was determined by inoculation at serial dilutions.

Before the start of the experiment, there were 5.4 ± 0.5 billion cells in capsules, 5.2 ± 0.5 billion cells when stored within 7 years, when stored at a temperature of minus 20 to 22 ° C 5.4 ± 0.6 billion cells, when stored at a temperature of 110 ° C, contain 5.3 ± 0.6 billion cells. The information obtained thus shows that the specified strain can be stored in a wide temperature range within about 7 years.

Examination of the properties of the new strain Bacillus subtilis 534 has shown that it is non-toxic, has no teratogenic and allergy effects.

During the cultivation, the strain allows the milk and milk products to clot by excreting proteolytic ferments. It also forms an antibacterial protein substance with a broad spectrum of activity and an immunomodulator.

The sour milk product that can be produced has a sweet taste and no smell. At a temperature between 2 and 8 ° C the product is stored within 7 days and above without loss of its organoleptic and therapeutic-prophylactic properties.

Attempts were made to enable the development of the foreign microflora in the manufacture of a milk product. 3 dm ^{3 of} milk were pasteurized at a temperature of 90 ° C. within 30 minutes. The milk was filled into 6 glass bottles of 0.5 dm ³ capacity. In each bottle, 1 cm ³ acid alarm clock was inserted, each containing 1 billion cells of the living parent culture Bacillus subtilis 534. The bottles were closed with cotton-gauze stoppers and placed in a thermostat where the temperature was kept at 37 ° C. After 2 hours, 2 samples were inoculated with 1 billion cells each from the living parent culture Staphylococcus aureus and 2 other samples with 1 billion cells each from the living parent culture Shigella flexneri. A uniform clot is formed in the experiment after 14 to 16 hours and in the control after 10 to 12 hours. In the test and control (milk product), only 1 strain of Bacillus subtilis 534 was inoculated.

To better understand the present invention, examples of the production of a sour milk product are given and cited its use.

example 1

50 dm ³ milk is pasteurized at a temperature of 75 ° C. within 30 minutes. Immediately after the pasteurization has been completed, the milk is filled into glass bottles with a capacity of 0.5 dm ³ . 1 cm ^{3 of} acid alarm clock is introduced into it, which contains 10 billion cells of the living parent culture Bacillus subtilis 534. The bottles are sealed with foil covers and placed in a thermostat at a temperature of 30 ° C. The even, dense clot is formed after 6 hours. A product is obtained which has a slightly sweet taste and a uniform consistency and no smell, and lacks the phosphatase. The acidity is 20 ° T. The contamination with the foreign microflora has not been proven.

The milk product produced was kept at a temperature of 8 ° C for 7 days without loss of its organoleptic properties. The acidity increased insignificantly to 28 ° T. The contamination (contamination) with the foreign microflora was not proven. Under the same conditions, 6 samples of kefir made using lactic acid bacteria were stored at the same time. The product became unusable after 48 hours.

Example 2

2 dm ³ buttermilk is pasteurized within 15 minutes at a temperature of 100 ° C. At the same time, it is inoculated with 4 cm ^{3 of an} alarm clock, which contains 500 cells of the living culture Bacillus subtilis 534, each 1 cm ³ .

The milk together with the alarm clock is placed in a thermostat, which maintains the temperature at 38 ° C. The complete fermentation takes place after 24 hours. A product is obtained which has a slightly sweet taste and no smell. The acidity is 49 ° T. The contamination with the foreign microflora has not been proven.

Example 3

Tests were carried out on the milk product produced according to Example 1. A sick person p. 11 months old. Diagnosis: diabetes, dysbacteria. Antibiotics were used for pneumonia. At the age of 6 months, meteorism and facial skin and body rash are registered. The dysbacteria was diagnosed when examining faecal samples. The patient was given the sour milk product (produced according to Example 1) 10 cm ³ twice a day on an empty stomach. The facial and body rash disappeared on the 7th to 8th day. The fecal examination on day 15 showed that the composition of the microflora is normal.

Example 4

A cure is made with the sour milk product produced according to Example 2. A patient P, 24 years old. Diagnosis: purulent wound on the right lower leg, gastric ulcer. The product is prescribed to be taken 200 cm ³ twice a day. After 2 days, the Bacillus subtilis 534 strain, together with Staphylococcus aureus, was detectable in wound secretion. The wound was cleared of pus and epithelized on day 11. The patient noted a reduced seizure intensity from hunger pain.

Example 5

The sour milk product prepared analogously to that described in Example 1 was administered in an amount of 10 to 250 cm ³ once or twice a day within 10 days for the purpose of prophylaxis to 4 patients who had been treated with antibiotics for a long time. The bacteriological fecal examination carried out on the 12th day showed that the composition of the microflora does not differ from the normal in all patients. There was also no clinical manifestation of the dysbacteria, while the difference in the degree of dysbacteria in 5 patients was diagnosed by the control group.

Dairy products in the same doses were given to treat dysbacteria in 2, diathesis in 3, and purulent wounds in 3 patients. (One patient was suffering gastric ulcer). Clinically, the pathological processes were abolished on the 2nd to 9th day of treatment in all 8 patients. The bacteriological faecal examination showed that the composition of the microflora becomes normal on the 12th day. No pathological reactions were found in 10 healthy volunteers who took the specified sour milk product in an amount of 250 cm ³ twice a day within 1 month.

Industrial applicability

The method according to the invention is used for the production of sour milk foods which have the therapeutically prophylactic properties which are used for the prophylaxis and treatment of bacterial infections, dysbacteria, diathesis and food allergy.

Claims (2)

1. A process for the production of sour milk products by fermentation of milk or milk products with the help of the living bacteria with the end product, characterized in that a strain of bacteria Bacillus subtilis 534 is used as living bacteria, which on 29.03.88 in the Allunions-- Culture collection of the Institute of Biochemistry and Physiology of Microorganisms of the Academy of Sciences of the USSR deposited and under number B 1666

is registered. 2. The method of claim 1, characterized featuring Ge that 534 in an amount of 10 ³ to _{10 le} ¹¹ for fermentation of the strain Bacillus subtilis - is added reproduced cells per 1 dm ³ milk or milk products.